1. Field of the Invention
The present invention relates to microscopy and, in particular, to apparatus, methods, and systems for resolving axial position of nanoscale objects, including but not limited to single molecules or other nanoscale structures.
We disclose a new technique, sometimes called standing wave axial nanometry (SWAN), to determine the axial location of single nanoscale fluorescent objects with sub-nm accuracy and 3.7 nanometer (nm) precision. A standing-wave excitation pattern is generated by positioning an Atomic Force Microscope (AFM) tip or other reflecting surface (e.g. mirror) over a focused laser beam. A fluorescent nanoparticle or a single fluorescent molecule is positioned within the standing-wave and its emission phase difference is used to measure its location along the optical axis. SWAN has an axial precision and localization accuracy that is superior to all previous optical methods. Unlike other approaches, SWAN does not require custom optics or specially engineered substrates, which makes it easy to use with biological samples and live cells. SWAN can be easily integrated with other super-resolution and super-accuracy techniques to image with nanometer resolution along the lateral and axial directions. Moreover, unlike most interference based techniques, where the interference pattern repeats itself and limits the working range on the order of 250 nm, successive periods can be distinguished in SWAN which extends the working distance of this technique. Another unique advantage of SWAN is that it can be used to determine the axial position of molecules in single molecule AFM force measurements and in Single Molecule Cut and Paste applications for the bottom-up assembly of nanostructures.
Unraveling the conformation and function of biomolecules at the nanometer scale requires localizing single molecules with high accuracy and measuring distances between them with high resolution. While single fluorescent dyes can be localized with nm accuracy in the lateral direction, improving resolutions along the optical axis is more challenging.
Here we describe a new technique standing wave axial nanometry (SWAN), to image the axial location of a single nanoscale fluorescent object with sub-nm accuracy and 3.7 nm precision. A standing wave, generated by positioning an Atomic Force Microscope (AFM) tip or other reflecting surface over a focused laser beam is used to excite fluorescence; axial position is determined from the phase of the emission intensity. We use SWAN to measure the orientation of single DNA molecules of different lengths, grafted on surfaces with different functionalities.
2. Related Art
Fluorescence imaging of nanoscale biological assemblies rely on localizing molecules with high accuracy and measuring distances between them with high resolution. However, the resolution of conventional fluorescence microscopes is limited by the diffraction of light: with a high numerical aperture objective and visible excitation, resolution is about 200 nm in the lateral direction and 500 nm along the optical axis.
A single fluorescent molecule can be localized with nanometer accuracy along the x- and y-axis by determining the centroid of its point spread functions (PSF)1, a technique known as fluorescence imaging with one nanometer accuracy (FIONA)2. This approach has also been used to resolve the lateral separation between two dyes of the same or of different colors within a diffraction-limited spot3-5. FIONA has been combined with the stochastic switching of single molecule fluorescence to obtain high-resolution images of microscopic biological objects such as cells, an approach alternatively known as stochastic optical reconstruction microscopy (STORM)6, photoactivated localization microscopy (PALM)7, and fluorescence photoactivated localization microscopy (FPALM)8. The lateral resolution of fluorescence imaging can also be improved by using stimulated emission depletion (STED) to narrow the effective width of the PSF9, 10.
Unlike imaging in the x- and y-direction, improving resolution and single molecule localization accuracy along the optical axis is more challenging11. In STORM experiments, the z-position of a single fluorophore can be determined with 50 nm resolution using a cylindrical lens to distort the shape of the PSF12; resolution can be further improved to 20 nm by sandwiching the sample between two opposing objectives13. Better resolution, down to 10 nm, can be achieved using interferometry as demonstrated in interferometric photoactivated localization microscopy (iPALM) and 4Pi-single marker switching microscopy (4Pi-SMS)14, 15; this however requires the use of custom optics in a complicated layout for interference detection. Alternatively, fluorescence interference-contrast microscopy (FLIC) has been used to determine the height of dye monolayers with nanometer accuracy16; these experiments however require multiple replicas of the sample deposited on patterned silicon oxide surfaces which limits its applicability in single molecule biological imaging. Fluorescence interference has also been used to monitor the movement of single motor proteins on microtubules17.
Therefore a need for improvement in the art exists.